The specialized literature provides reliable data that long-term and distinctly elevated concentrations of prostaglandin E2 (PGE2) and nitric oxide (NO) appear with many serious diseases. Although their precise role in the etiopathogenesis of the diseases has not been defined as yet, the experimental and clinical findings so far document that both PGE2 and NO are rightfully the targets in the development of new medicaments.
PGE2 is a biologically active prostanoid produced by a multiple-step enzymatic conversion of arachidonic acid, which is contained in cell membranes. The final and key role in the formation of PGE2 is played by cyclooxygenase-2 (COX-2). Under normal circumstances, the activity of this protein in tissues and cells is negligible. A rapid rise of activity occurs under pathological conditions in reaction to various impulses of biological, chemical or physical nature. Pro-inflammatory cytokines, particularly interleukin-1β (IL-1β), the tumor-necrosis factor (TNF-α) and interferon-γ (IFN-γ) are significant activators [see e.g. Arterioscler. Thromb. Vasc. Biol. 20, 677-682, 2000 and Clin. Exp. Allergy 30, 1275-1284, 2000], as well as infections and UV radiation.
NO is a product of the conversion of the amino acid L-arginine by the enzyme NO synthase (NOS). There are three isoforms of this enzyme. Two of them (endothelial and neuronal NOS; i.e., eNOS and nNOS) produce constitutively very small amounts of NO. Their function is the regulation of the vascular tone and neurotransmission. Inducible NOS (iNOS) is found in almost all cells and tissues of the organism but under normal circumstances does not show any activity. Like in the case of COX-2, iNOS is activated, and consequently a very intensive production of NO occurs, under various pathological conditions, e.g., during hypoxia. The most important iNOS-activity stimulators are bacterial products (lipopolysaccharide, lipoteichoic acid, peptidoglycans) and some cytokines. NO has a fundamental importance in immune protection against viruses, bacteria and other parasites, but the damage of regulation processes resulting from permanent iNOS activation under pathological conditions, and hence from the long-term overproduction of NO, has very adverse consequences for the organism. Increased concentrations cause fatal hypotension and participate in the etiopathogenesis of especially inflammatory and cancerous diseases. Gaseous NO is unstable and easily transformed into toxic products such as peroxynitrite and others. A long-term increase in NO production subsequently leads to DNA damage.
Very effective inhibitors of COX-2 activity are glucocorticoids, which are however strongly immunosuppressive. In practice, both non-selective (ibuprofen, indomethacin) and selective COX-2 inhibitors, e.g., celecoxib, refecoxib, valdecoxib, parecoxib, etoricoxib, lumiracoxib (so-called ‘coxibs’), are used. They allow a relatively effective treatment of inflammatory diseases, predominantly of rheumatoid arthritis and osteoarthritis. However, they show undesirable side effects on the cardiovascular system, such as those discovered in rofecoxib, which was the reason for its withdrawal from the pharmaceutical market.
PGE2 is considered to be the cause of pain and fevers related to the process of inflammation. It is assumed that COX-2 activity and the increased PGE2 production play an important role also in the pathogenesis of neurodegenerative diseases with an inflammatory component [J. Mol. Neurosci. 33, 94-99, 2007]. Selective COX-2 inhibitors therefore reduce the risk of Alzheimer's [Brain 131, 651-664, 2008] and Parkinson's diseases and probably also of asthma. Likewise atherosclerosis is connected with the increased levels of PGE2, but in this case the administration of the selective COX-2 inhibitors known so far is not recommended due to the above-mentioned cardiotoxicity, and it is also for this reason that new types of inhibitors are currently being sought [Curr. Drug Targets Cardiovasc. Haematol. Disord. 5, 303-311, 2005].
The inhibition of PGE2 is considered to be one of the very prospective approaches not only with arthritic diseases but also in tumor-disease therapy [W. K. Wu, J. J. Yiu Sung, C. W. Lee, J. Yu, C. H. Cho, Cancer Lett. 2010, electronically published before its publication in print]. The increased activity of the COX-2 enzyme and the excessive production of PGE2 were found in tumors of the large intestine [Gastroenterology 107, 1183-1188, 1994], stomach [Cancer Res. 57, 1276-1280, 1997], lungs [Cancer Res. 58, 3761-3764, 1998] and breast [Int. J. Oncol. 10, 503-507, 1997]. Of the mechanisms which participate in the procancerogenous effect of PGE2, antiapoptic and angiogenic effects have been described [J. Cancer Res. Clin. Oncol. 127, 411-417, 2001]. In connection with a possible antitumor use of the PGE2-production inhibitors, tumors of the large intestine are most often considered [Biochim. Biophys. Acta 1766, 104-119, 2006]; but their applicability may be wider [Oncogene 29, 781-788, 2010]. COX-2 inhibition reduces, e.g., the risk of the formation of non-melanoma skin tumors after UV irradiation [Photochem. Photobiol. 84, 322-329, 2008]. Two of the COX-2 inhibitors, celecoxib and rofecoxib, have been authorized by the FDA as supplements in the standard treatment of patients with familial adenomatous polyposis [Front. Biosci. 9, 2697-2713, 2004].
According to recent, experimentally substantiated, results it is expected that the treatment of inflammatory and cancer diseases is more effective when the PGE2 inhibitors are administered simultaneously with NO inhibitors, although both show antitumor effects already on their own. For instance, the selective COX-2 inhibitor nimesulide and iNOS inhibitor L-NG-nitroarginine reduce the carcinoma of the large intestine in sewer rats [Biofactors 12, 129-133, 2000]. The maximum protective effect against the development of experimental ulcerative colitis in sewer rats was described under simultaneous administration of the COX-2 inhibitor rofecoxib and the iNOS inhibitor aminoguanidine [Inflammopharmacology 15, 188-195, 2007]. The simultaneous inhibitory activity of melatonine on NO and PGE2 production is also considered to be the most likely mechanism of its positive effect on colitis in experimental animals [World J. Gastroenterol. 9, 1307-1311, 2003].
The PGE2 inhibitor (celecoxib) and also selective iNOS inhibitors (aminoguanidine and SC-51) reduce the development of the tumor of large intestine, experimentally induced in sewer rats. The antitumor efficiency distinctly increases when both types of inhibitors are administered at the same time [Cancer Res. 62, 165-170, 2002]. The simultaneous inhibitory effect on NO and PGE2 production is used to explain also the antitumor effects of some substances of natural origin, e.g., obtained from rubus occidentalis (blackberry) [Cancer Res. 66, 2853-2859, 2006].
Within the scope of the present invention, it was discovered that novel 5-substituted pyrimidine derivatives are able to provide a dual, or simultaneous, reduction of NO and PGE2 production, and thus they can be used for the treatment of inflammatory and cancer diseases.
Substituted pyrimidines are substances well known from the literature [e.g., the synoptic review: Rewcastle, G. W. Pyrimidines and their Benzo Derivatives; Comprehensive Heterocyclic Chemistry III, 2008, 8, 117-272. Elsevier, Oxford]. For their preparation, 2,4-dihalogenopyrimidines are often used, the halogen atoms of which are subsequently modified by means of a wide range of reactions. These 2,4-dihalogenopyrimidines were studied predominantly as intermediates in the preparation of other substituted pyrimidines, mostly without their biological activity being tested. The antiviral activity of 2-amino-4,6-dichloropyrimidine is known in the literature [Annals of the New York Academy of Sciences 284, 294-304, 1977; Experientia 35(3), 321-322, 1979]. Some works have even dealt with the testing of the anti-inflammatory activities of pyrimidine derivatives, but actual 4,6-dihalogenopyrimidines had been considered to be intermediates only and their biological activity has therefore not been studied [{hacek over (C)}eskoslovenská farmacie 10, 433-439, 1986]. Furthermore, the halogen atoms in positions 4 and 6 offer the possibility to prepare the corresponding mono- or diarylpyrimidines using methods described in the literature [Journal of Medicinal Chemistry 50, 2060-2066, 2007; Journal of Heterocyclic Chemistry 46, 960, 2009].
5-Substituted 4,6-dihalogenopyrimidines are very little known from the literature. The exception is 2,5-diamino-4,6-dichloropyrimidine which is abundantly used as an intermediate product in the preparation of purine derivatives [e.g. Nucleosides, Nucleotides & Nucleic Acids, 19(1.2), 297-327, 2000].
The following further examples of the use of 5-substituted pyrimidine derivatives are known from the literature:                1) The compounds of the following formula as glycogen synthase kinase (type 3) inhibitors—this enzyme is one of the main regulating enzymes of glycogen turnover WO 2007/040440]:        
                 wherein R8 and R9 are only H, CN and halogens.        2) The compounds of the following formula as non-selective inhibitors of the formation of a wide range of cytokines such as TNF-α, IL-1, IL-6, IL-1β, IL-8, IL-12 and as non-selective inhibitors of a wide range of enzymes such as thromboxane-synthase and cyclooxygenase (types 1, 2 and 3):        
                 wherein R5 can only be hydrogen, —OH, —NH2, —N3, alkyl, alkyloxy, aryloxy, heteroaryloxy, —SR6, —S(O)nR7, haloalkyl, aminocycloalkyl, aminoalkyl, aminodialkyl, —NH(C1-C5)nX, —NH(CH2)nOH, —NHNH2 and alkylhydrazines.        
The substances of this formula show a very high toxicity in the concentrations used for determining the production of cytokines [WO 2007/031829] and an essential part of their effect can thus be solely cytocidal effect on the cells of the immunity system. Such substances have practically no therapeutic potential.                3) The compounds of the following formula as strong cancerostatics [WO 2006/079556]. These compounds are highly cytotoxic in nanomolar concentrations:        
                 wherein X can only be NR1R2, OR or SR.        4) The compounds of the following formula as substances stimulating the immunity system through an interaction with the TLR 7 receptor [WO 2009/067081]:        
                 wherein R1 can only be alkyl, alkoxy, alkylthio and wherein R3 is only hydrogen or alkyl.        5) The compounds of the following formula as substances inhibiting a wide range of kinases and phosphatases with a cytotoxic effect for use as cancerostatics [US 2009/0318446]:        
                 wherein R3 and R4 are any hydrocarbon substituents or OR, COR, COOR, CN, CONR1R2, NR1R2, SR, SOR, SO2R, SO2NR1R2, R, halogen, CF3, NO2 or an alicyclic substituent. All substances contain an indole ring connected directly to the pyrimidine ring.        6) The compounds of the following formula as substances inhibiting phosphoinositide-3-kinase (PI3Ks) with a cytotoxic effect for use as cancerostatics [WO 2009/120094]:        
                 wherein R1 and R2 are independently aryl, heteroaryl or heterocycloalkyl; R5 is only halogen or —OSO2R; Q is any linker; T is only —CO—, —CS—, —SO2—; X, Y and Z are independently nitrogen or CR, R is hydrogen or a lower alkyl.         The necessary pre-requisite for the biological activities of these substances is the presence of the chemically reactive alkylation group T-C—R5.        7) The compounds of the following formula as substances inhibiting (protein-tyrosin)kinase for use as cancerostatics [WO 2006/000420]:        
                 wherein R1, R2, R3, R4 and R5 are almost any substituent; X, Y and Z are independently nitrogen or CR5. Among the substituents X and Y, however, only an arbitrarily substituted phenylaminocarbonylamino group is possible.        8) The compounds of the following formula as substances inhibiting HMG-CoA-reductase, thereby reducing the speed of cholesterol biosynthesis [WO 2005/030758]:        
                 wherein R1, R2, R3 and R4 are almost any substituents; X is nitrogen or CR5; Z can only be fragments corresponding to these formulas:        
                9) The compounds of the following formula as substances inhibiting the production of IL-1, IL-6, IL-8, TNF-α and TNF-β for the treatment of diseases caused by these cytokines [US 2000/006096748]:        
                 wherein R3 and R4 can only be NR5R6, NHS(O)2R7, NR10C(Z)R8, NR10C(Z)NR5R6, NR10C(═NR11)OR10 or NR10C(Z)NR5R6.        10) The compounds of the following formula as stimulators of the production of a nerve-growth factor for the treatment of neurodegenerative diseases [WO 99/19305]:        
                 wherein R1 can only be an amino group substituted by one or two alkyl residues, which may be further substituted. These alkyl residues may even jointly form a ring, but this ring can only be heterocycloalkyl.        11) The compounds of the following formula for use as cancerostatics [CA 2093203]:        
                 wherein R2 and R3 can only be hydrogen or a lower alkyl.        12) The compounds of the following formula as inhibitors of transcription factor activation (such as NF-κB and AP-1) for use as anti-inflammatory medicaments [U.S. Pat. No. 5,811,428]:        
                 wherein R2, R4 and R6 are almost any substituents and R5 can only be —C(O)NRaRb, —C(S)NRaRb, —NRaC(O)Rb and —NRaC(S)Rb.        
These substances inhibit the production of a wide range of cytokines and other signal molecules, such as IL-1, IL-2, IL-8, TNF-α, TAP-1, MHC, E-selectin, VCAM-1, ICAM-1, c-mys, ras and p53. These factors (NF-κB and AP-1) also have further natural biological functions such as the participation of NF-κB in the transfer of the nerve signal (synaptic plasticity) or memory storage [Synapse 35 (2), 151-159, 2000]. The above-mentioned substances with a non-selective effect have little therapeutic potential.